Purification of Alkaloids with RediSep® Rf SCX Columns

By Teledyne ISCO

Abstract: The purification of alkaloids using RediSep Strong Cation Exchange (SCX) columns is described. Several alkaloids are used as models for those extracted from plant species. The alkaloids are separated from neutral and acidic compounds, and are purified from each other. Solvent switching is used to purify increasingly basic compounds.

Background: Why use SCX?

Ion exchange media separate molecules by net charge. Because ions have differing affinities for the ion exchange media, it is possible to selectively remove ions from solutions and release them later. SCX columns can be used in a “catch-andrelease” mechanism where the basic compounds are removed from the crude mixture and released after the impurities are washed away. Alternatively, the ionic strength and pH of the solvent system can be altered to purify a collection of bases. Both methods were combined to isolate alkaloids from green tea extract.

RediSep Rf SCX columns can be used with solvents such as dichloromethane.

Experimental and Results

Compounds were dissolved in acidic methanol as dissolution in neutral solvent failed to bind the compound to the column.

Harmaline was not displaced by the hydrogen ion, but required the ammonium ion
to displace the alkaloid.

Green Tea Extract

Green tea extract is used as a model to demonstrate the technique using an actual plant extract. Green tea was extracted in methanol and the extract dried. A portion of this extract (0.5 g) was dissolved in 5 mL methanol containing 5% glacial acetic acid; the entire sample was injected onto a 15 g RediSep Rf SCX column. Detection was at 210 and 270 nm. A gradient using 5% acetic acid in water, followed by a gradient containing 5% ammonium hydroxide in water was used to isolate the alkaloids.

The non-polar and acidic compounds in the plant extract are washed off the column early in Figure 3. The alkaloids elute as the acetic acid gradient is run. Other basic compounds elute during the second gradient with ammonium hydroxide. The last gradient regenerates the column for subsequent use. A time window was used so that fractions were only collected during the first two gradients and not during column regeneration.

Acetic acid and ammonium hydroxide are used because they are easily removed from the sample after purification.


Teledyne Isco RediSep Rf SCX columns are useful for isolating basic compounds. The columns can be used for “catch-and-release,” or to purify a mixture of basic compounds. RediSep Rf SCX columns are useful for purifying alkaloids from complex mixtures. Several alkaloids were captured and released, suggesting this technique to be general. Dissolving the alkaloids in an acid solution causes them to be cationic for capture.

Additional SCX column experiments are highlighted in Teledyne Isco’s Application Note 40 available at www.isco.com/lcappnotes.

Published In

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Go, Go Gadgets

Published: July 5, 2011

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